Cyclic AMP (cAMP) is one of the fundamental signaling molecules used by colonic epithelial cells to control basic cellular functions (e.g. proliferation, apoptosis, differentiation, fluid secretion and polarization) that ultimately contribute to the function and integrity of the mucosal barrier. Normally, cAMP signals arise through activation of cell surface G-protein coupled receptors (GPCRs), a process that is tightly regulated. However, findings made during the prior funding period led to the identification of a previously unknown signaling pathway that connects the endoplasmic reticulum (ER) calcium sensor STIM1 to the production of cAMP in colon crypt- derived cells. Similar to store-operated or capacitative Ca2+ entry, this GPCR-independent cAMP signaling pathway is directly activated by any maneuver that causes free [Ca2+] within the ER lumen to become reduced, and is absolutely independent of cytosolic [Ca2+]. It requires translocation and clustering of STIM1 from the bulk ER to sites closely apposed to the plasma membrane, resulting in activation of conventional transmembrane adenylyl cyclases by an unknown process. Our overall hypothesis is that this new store-operated avenue to cAMP production represents a protective mechanism that becomes engaged following non- physiological, catastrophic loss of the ER Ca2+ store. The ensuing cAMP elevation triggers adaptive responses, including cAMP-stimulated ion and fluid secretion. Over the long term, however, inappropriate or persistent elevation of cAMP is expected to cause alterations in gene expression and cell division that may ultimately disrupt the function of the epithelium, or even elicit adenoma formation. Here we propose to determine the domains within STIM1 that are responsible for activation of adenylyl cyclase following depletion of ER Ca2+ stores (Specific Aim #1). For this purpose we will use molecular approaches for altering the STIM1 protein and sensitive FRET-based reporters for imaging cAMP to follow the effects of mutated STIM1 in single NCM460 colonic epithelial cells. We will then investigate how phosphorylation of STIM1 regulates this pathway (Specific Aim #2). In Specific Aim #3 we will identify other elements of the store-operated cAMP signaling cascade using a genome-wide RNAi library to knock down individual gene candidates. For this purpose our lab has developed a high-throughput functional screen of cAMP dynamics that can be performed in live cells using FRET-based cAMP reporters. Finally, we propose to investigate the extent to which store depletion-dependent cAMP generation regulates the ion transport properties of the native epithelium of the rat large intestine (Specific Aim #4). Potential Impact on Veteran<s Health Care: Diseases of the colon, including colon cancer, are pervasive health issues in the aging veteran population. The store- operated pathway described may be subject to activation by constituents in the colonic lumen, such as bile acids, metabolites of commensal bacteria, ingested drugs and pathogens (viral and bacterial) that can cause non-physiological release of the ER Ca2+ store. Because cAMP influences proliferation, apoptosis, differentiation, fluid secretion and polarization of the crypt cell, the ensuing cAMP signals are expected to impact wide-ranging aspects of digestive physiology and barrier function, including control of the maturation of epithelial cells along the crypt-villus axis and fluid secretory activity that underlies diarrhea.